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CN110592209A - Method and kit for detecting polymorphism sites of cardiovascular and cerebrovascular drug-related genes - Google Patents

Method and kit for detecting polymorphism sites of cardiovascular and cerebrovascular drug-related genes Download PDF

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CN110592209A
CN110592209A CN201910975111.7A CN201910975111A CN110592209A CN 110592209 A CN110592209 A CN 110592209A CN 201910975111 A CN201910975111 A CN 201910975111A CN 110592209 A CN110592209 A CN 110592209A
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extension
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董新波
卜令斌
张瑞
刘秀财
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Koran Medical Laboratory Xuzhou Co Ltd
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Abstract

The invention discloses a detection method and a kit for cardiovascular and cerebrovascular drug-related gene polymorphic sites, and belongs to the technical field of molecular biology. Compared with the first-generation sequencing, the detection method and the detection kit have the advantages that the rapid turnaround time of one day is shorter; the reagent material consumption is simple, the experiment is operated in a flow process, and expensive reagents such as fluorescent dyes or probes are not needed; because the mass spectrometry technology directly detects the molecular weight (mass-to-charge ratio) of DNA, theoretically, only one copy of amplified DNA fragment can be amplified for identification, thereby having high sensitivity and smaller deviation compared with RT-PCR; the reaction can be carried out in a micro system, the capability of high multiplicity can be realized in a single reaction hole, the detection capability can reach 40 weight at most, and the detection efficiency is improved.

Description

Method and kit for detecting polymorphism sites of cardiovascular and cerebrovascular drug-related genes
Technical Field
The invention relates to a detection method and a kit for cardiovascular and cerebrovascular drug related gene polymorphic sites, belonging to the technical field of molecular biology.
Background
Genetic variations in the metabolism and transport of drugs in vivo and changes in their expression levels affect the in vivo concentration and sensitivity of drugs, resulting in drug-responsive individual differences. With the continuous development of gene detection technology in recent years, the detection of the drug genome is more and more accurate and more convenient. Pharmacogenomics has become an important tool for guiding clinical individualized medication, evaluating the risk of serious adverse drug reactions and guiding the research, development and evaluation of new drugs. Currently, molecular detection of drug response-related genes and their expression products is a prerequisite for the implementation of personalized drug therapy.
The individual antihypertensive drug administration instruction: 1) diuretics: hydrochlorothiazide, furosemide, torasemide, bumetanide
According to The Genetical and genomic pharmacological databases (PharmGKB-The Pharmacogenetics)&Pharmacogenomics knowledgbase) suggested that the ADD1 gene correlates with the efficacy of diuretics (grade: 2B) in that respect ADD1 is an α/β heterodimeric skeletal myomembrane protein that affects intracellular sodium-potassium pump function. The gene is mutated to enhance Na+-K+The activity of ATPase, increasing the reabsorption of sodium by the renal tubules and increasing blood pressure, thus affecting the diuretic effect.
2) Calcium ion antagonists: verapamil and nifedipine
According to The guidance of The Pharmacogenomics and genomics databases (PharmGKB-The Pharmacogenetics & Pharmacogenetics knowledgebase), The CACNA1C gene was associated with The efficacy of calcium ion antagonists (grade: 3). The alpha 1C subtype of the L-type calcium ion channel alpha 1 subunit is a main action target spot of dihydropyridine calcium ion channel blockers for regulating cardiovascular functions and reducing blood pressure, and the gene CACNA1C SNPs of the L-type calcium ion channel alpha 1 subunit may have a certain relation with the blood pressure reducing effect of the calcium ion channel blockers.
According to The guidance of The pharmacogenetic and genomics databases (PharmGKB-The Pharmacogenetics & Pharmacogenetics knowledgebase), The CACNA1C gene was associated with The therapeutic effect of nifedipine (grade: 3). CYP3a5 is involved in the metabolism of various drugs. The CYP3A5 gene 3 intron 3 contains mutation of 6986A > G (rs776746, CYP3A 5. multidot.3) at position 22893, the SNP can cause abnormal splicing of CYP3A5mRNA, and causes premature cutting of CYP3A5 protein by a stop codon, so that the CYP3A 5. multidot.3 homozygous individual liver and CYP intestinal tract 3A5 protein expression and activity are obviously reduced. Significant ethnic differences exist in CYP3A5 x 1 allele frequencies, ranging from 10% to 15% in white people, 28% in Chinese people and up to 60% to 80% in black people. The gene mutation causes the slow drug metabolism rate, the increase of blood drug concentration and the enhancement of toxic and side effects, and the drug dosage is recommended to be reduced as appropriate.
3) Beta receptor blockers: atenolol, metoprolol, carvedilol
According to The guidance of The Pharmacogenomics and genomics databases (PharmGKB-The Pharmacogenetics & Pharmacogenetics knowledgebase), The CACNA1C gene was associated with The efficacy of atenolol (grade: 3). The gene mutation can increase the sensitivity of individual to medicine and enhance the curative effect of medicine.
According to the guidance in technical guidelines for detecting genes for drug metabolizing enzymes and drug target of action (trial), it is suggested that beta-adrenoceptor (beta-adrenoceptor) is a subfamily of adrenoceptors, belonging to the superfamily of G protein-coupled receptors, including beta-adrenoceptors1、β2And beta3Three different subtypes. The receptor regulates cAMP and L type Ca in cells by coupling with Gs protein2+The opening frequency of the channel is the target of the action of beta receptor agonist and beta receptor blocker. Beta is a1Polymorphism in the receptor encoding gene ADRB1 can affect the efficacy of beta receptor blockers such as metoprolol. The ADRB1Gly389Arg (rs1801253) polymorphism results in two types of receptors at positions Arg389 and Gly389, wherein the coupling efficiency of the Arg389 type receptor to the G protein is higher than that of the Gly389 type receptor. The degree of blood pressure reduction of an Arg389 homozygote hypertensive patient after metoprolol application is 3 times that of an individual with a Gly389Arg heterozygote genotype; the improvement of left ventricular ejection fraction is better after the Arg389 homozygote genotype heart failure patient is treated by carvedilol and metoprolol. The clinician is advised to apply beta1ADRB1 polymorphism detection is carried out before receptor blocking drugs, and the dosage is adjusted according to the genotype of the receptor blocking drugs, so that the curative effect is improved, and the occurrence of adverse reactions is reduced.
According to the guidance in technical guidelines (trial) for the detection of drug metabolizing enzymes and drug action target genes, CYP2D6 is also known as isoquinoidine 4' -hydroxylase, an important member of the CYP second subfamily. The CYP2D6 activity in the population is manifested by a four-state distribution of strong (EM), Intermediate (IM), weak (PM) and ultra-strong (UM) metabolites. The incidence of CYP2D6PM in caucasian population is as high as 5-10%, while the incidence of PM in oriental population is about 1%. Polymorphisms that result in the loss of CYP2D6 enzyme activity can affect the in vivo metabolism of antipyrine, codeine, beta blockers such as metoprolol and carvedilol, chlorimipramine, nortriptyline, desipramine, doxepin, imipramine, maprotiline, opipramol, trimipramine, ondansetron, tramadol, and tamoxifen, and so on, thereby affecting the efficacy and adverse effects of these drugs, and requiring dose adjustments based on individual genotypes for clinical use.
4) Angiotensin ii receptor antagonists: losartan, valsartan, olmesartan
According to the guidance in technical guidelines for the detection of genes for drug metabolizing enzymes and drug targets (trial), CYP2C9 is an important member of the second subfamily of cytochrome P450 enzymes (CYP), accounting for 20% of the total amount of liver microsomal P450 protein. CYP2C9 participates in the hydroxylation metabolism of various drugs such as anticoagulants, anticonvulsants, hypoglycemic drugs, nonsteroidal antipyretic analgesic and anti-inflammatory drugs, antihypertensive drugs, diuretic drugs and the like. Changes in CYP2C9 activity can lead to large changes in the in vivo concentration of these drugs and even to the occurrence of serious adverse drug reactions. Losartan is a commonly used antihypertensive drug, and is metabolized and activated in vivo mainly through CYP2C9 to be a metabolite E-3174 with the antihypertensive effect. Individuals carrying the CYP2C9 x 3 allele have a reduced production of E-3174 and a reduced metabolic rate of losartan after administration of losartan. After 1h to 6h after the oral administration of the losartan with single dose, the blood pressure reducing effect of the losartan in individuals with CYP2C9 x 1/x 3 genotypes is reduced, and the dosage of the losartan needs to be properly increased so as to enhance the blood pressure reducing effect.
According to the guidance and suggestion in technical guidelines for gene detection of drug metabolizing enzymes and drug action targets, organic anion transport polypeptide 1B1(OATP1B1, also known as OATP-C, OATP2 or LST1) is specifically expressed on a hepatocyte basement membrane, and plays an important role in the uptake and elimination of endogenous and exogenous substances such as statins, repaglinide, enalaprilat, temocapril, valsartan, olmesartan and the like by hepatocytes. OATP1B1 is coded by SLCO1B1 gene, the polymorphism of exon 521T > C (Val174Ala) 5 of the gene is the main genetic variation in Asian population, the allele frequency is 10-15%, and the polymorphism can obviously reduce the substrate uptake capacity of OATP1B1 and increase the blood concentration.
5) Angiotensin converting enzyme inhibitor: fosinopril, enalapril, lisinopril, captopril, temocapril
According to guidance suggestions in technical guidelines for detecting drug metabolism enzymes and drug action target genes (trial) issued by the Weitexus, ACE I/D polymorphism can influence the ACE level of plasma, the ACE activity of individual with DD genotype is increased, and the ACE activity is reduced more obviously after enalapril treatment; among the hypertension patients treated at first, fosinopril of the DD type patients has enhanced antihypertensive effect; in patients with hypertension combined with left ventricular hypertrophy and diastolic filling disorder, the degree of improvement of cardiac function of patients with DD genotype is better than that of patients with ID and II genotypes after taking enalapril and lisinopril; the renal function of patients with genotype II is more significantly reduced by lisinopril or captopril. In order to obtain the best curative effect, the detection of ACEI/D polymorphism is recommended to be clinically carried out before ACEI medicines are selected for treatment so as to guide the selection of proper ACEI medicines.
However, the existing molecular detection method mainly comprises restriction fragment length polymorphism analysis, PCR sequencing and gene chip, and generally has the defects of low flux, low resolution, high background signal, high cost and the like.
The MassArray molecular weight array technology is the leading gene mutation analysis technology in the world. MassArray combines simple and reliable primer extension reaction and advanced MALDI-TOF mass spectrometry technology, can quickly and economically perform genotype analysis, and achieves the highest accuracy and cost performance in the current market. The iPLEX GOLD technology based on the MassArray molecular weight array platform can conveniently design up to 40 genotypes for detection. The experimental design is flexible and the price is low.
Disclosure of Invention
In view of the problems of the prior art, the first objective of the present invention is to provide a method for detecting the polymorphic sites of the cardiovascular drug-related genes.
The second purpose of the invention is to provide a detection kit for the polymorphic sites of the cardiovascular and cerebrovascular drug related genes.
In order to achieve the above object, the present invention provides a method for detecting a polymorphic site of a cardiovascular and cerebrovascular drug-related gene, wherein the polymorphic site of the cardiovascular and cerebrovascular drug-related gene comprises at least one of ADD1-RS4961, CYP2C 9-3-RS 1057910, CACNA1C-RS1051375, CYP2D 6-10-RS 1065852, SLCO1B1-RS4149056, CYP3a5-RS776746, ADRB1-RS 1801253; preferably, the cardiovascular and cerebrovascular drug related gene polymorphism site consists of ADD1-RS4961, CYP2C 9-RS 1057910, CACNA1C-RS1051375, CYP2D 6-RS 1065852, SLCO1B1-RS4149056, CYP3A5-RS776746, ADRB1-RS 1801253;
the detection method comprises the following steps:
1) preparation of PCR amplification primers: the PCR amplification primer is designed according to the selected individualized medication related gene to be detected, and is an amplification primer specific to each gene, wherein the amplification primer can amplify a DNA sequence including a mutation site, the amplification primer (including a forward primer and a reverse primer) has 17-25 bases which are completely matched with the gene sequence to be targeted at the 3' end, and has a tag sequence at the 5' end, preferably has the same tag sequence at the 5' end, and more preferably has the tag sequence of acgttggatg so as to distinguish the amplification primer from an extension primer;
wherein, the amplification primer comprises at least one pair of SEQ ID NO 1-14 sequences, preferably the amplification primer comprises SEQ ID NO 1-14 sequences;
2) preparation of mass spectrum extension primer: the length of the mass spectrum extension primer is 15-28 bases, the 3' end of the mass spectrum extension primer is located at the last base of the mutation site of the related gene for personalized medicine in the extension direction, the extension primer only extends one base when in extension reaction, and the extended base is the mutation site of the related gene for personalized medicine;
wherein the difference in molecular weight between the extension product and the extension primer is not less than 9 Da; primers were designed according to the principle that no dimer was formed between the primers, no hairpin structure was formed between the primers and the amplification product/extension product, and no mismatch occurred between the primers and the template, and in particular, the extension primers for mass spectrometric detection were designed according to the following principle: the molecular weight difference of each extension product is not less than 30Da, the molecular weight difference between each extension primer and each extension product is not less than 9Da, and the molecular weight of the extension primer and the extension product is between 4500-8500 Da;
wherein the extension primer comprises at least one pair of SEQ ID NO 15-21 sequences, preferably the extension primer comprises SEQ ID NO 15-21 sequences;
3) preparation of a detection template: extracting DNA of a sample to be detected by adopting a proper commercial kit, wherein a Tiangen genome DNA extraction kit is adopted for clinical samples;
4) performing PCR amplification by using the DNA extracted in the step 3) as a template and the PCR amplification primer in the step 1) to obtain a target sequence amplification product;
5) removing unreacted dNTPs contained in the amplification product obtained in the step 4) by SAP enzyme treatment;
6) using the amplification product obtained in the step 5) as a template, and connecting a base to the 3' end of the extension primer through an extension reaction by using the mass spectrum extension primer in the step 2), thereby obtaining an extension product;
7) purifying the extension product obtained in step 6);
8) and (3) carrying out mass spectrum detection on the purified product on a mass spectrometer, and analyzing the obtained mass spectrum peak diagram through analysis software to obtain a typing result.
In addition, the invention also provides a detection kit for the cardiovascular and cerebrovascular drug related gene polymorphic sites, wherein the kit comprises a PCR amplification primer and a mass spectrum extension primer;
the PCR amplification primer is designed according to the selected individualized medication related gene to be detected, and is an amplification primer specific to each gene, the amplification primer can amplify a DNA sequence including a mutation site, the amplification primer (including a forward primer and a reverse primer) has 17-25 bases which are completely matched with the gene sequence to be targeted at the 3' end, and has a tag sequence at the 5' end, preferably has the same tag sequence at the 5' end, and more preferably has the tag sequence of acgttggatg, so as to distinguish the amplification primer from an extension primer;
wherein, the amplification primer comprises at least one pair of SEQ ID NO 1-14 sequences, preferably the amplification primer comprises SEQ ID NO 1-14 sequences;
the length of the mass spectrum extension primer is 15-28 bases, the 3' end of the mass spectrum extension primer is located at the last base in the extension direction of the mutation site of the related gene for personalized medicine, the extension primer only extends one base when extension reaction occurs, and the extended base is the mutation site of the related gene for personalized medicine;
the extension primer comprises at least one pair of sequences from SEQ ID NO. 15 to SEQ ID NO. 21, and preferably, the extension primer consists of SEQ ID NO. 15 to SEQ ID NO. 21.
Compared with the prior art, the invention has the beneficial effects that:
1) the reagent consumables used in the invention are relatively simple and stable in performance, and do not need expensive reagents such as fluorescent dyes and special enzymes.
2) The reaction can be carried out in a micro system, and the use of samples and various consumables is reduced.
3) Because the mass spectrometry technology directly detects the molecular weight (mass-to-charge ratio) of DNA and directly determines the type of a base (i.e. does not need to undergo any form of signal conversion), theoretically only one copy of amplified DNA fragment can be identified by amplification, and thus, the sensitivity is high; the mass spectrometry technology has the characteristics of automation, high-throughput detection and the like, so that the mass spectrometry technology is combined with a multi-primer extension technology, and a plurality of drug-taking related sites can be simultaneously detected in one reaction system, thereby greatly reducing the workload and improving the detection throughput.
4) The invention adopts a flight time mass spectrum genotyping system, realizes the detection and the genotyping of the individualized medication related genes of the medicines by designing a set of brand new mass spectrum extension primers, can simultaneously and accurately detect the correlation of the polymorphic sites of the cardiovascular and cerebrovascular medication related genes compared with other methods, and has the remarkable advantages of high throughput, automation, low cost and the like.
Drawings
FIG. 1 is a mass spectrum peak diagram of ADD1-rs4961 detected by using DNA extracted from a sample as a template, and the analysis result is homozygous G, which shows that the curative effect on diuretic drugs is normal.
FIG. 2 is a mass spectrum peak diagram of ADD1-rs4961 detected by using DNA extracted from a sample as a template, and the analysis result is homozygous GT, which shows that the curative effect on diuretic drugs is enhanced.
FIG. 3 is a mass spectrum peak diagram of ADD1-rs4961 test using DNA extracted from sample as template, the analysis result is homozygous G, showing enhanced therapeutic effect on diuretic drugs.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention.
Firstly, the detection kit for the cardiovascular and cerebrovascular medication related gene polymorphic sites comprises:
1) PCR reaction Mix: including 1 XPCR reaction buffer (purchased from Agena biosciences), the PCR amplification primers (biosynthesized by Shanghai Biotechnology);
2) PCR reaction Taq enzyme (available from Agena Bioscience);
3) SAP reaction buffer (available from Agena Bioscience);
4) SAP enzyme (available from Agena Bioscience);
5) extension reaction solution Mix: including reaction buffer (purchased from agena bioscience), the mass spectrometry extension primer (biosynthesized by shanghai life);
6) extension reaction Taq enzyme (available from Agena Bioscience, Inc.);
7) MassARRAY chip (from Agena Bioscience).
In the following examples, unless otherwise specified, all the methods are conventional.
The primer sequence is biosynthesized by Shanghai Biotechnology, PCR amplification reagents, SAP enzyme, Extension reaction liquid and MassARRAY chip are purchased from Agena Bioscience, a PCR amplification instrument is model 9700 of the AB company, and a mass spectrum Analyzer is MassARRAY Compact Analyzer of the Agena Bioscience.
Example 1
A detection method of the polymorphism sites of the cardiovascular and cerebrovascular medication related genes comprises the following steps:
1) preparation of PCR amplification primers: designing an amplification primer specifically aiming at each individualized medication related gene according to the selected individualized medication related genes of the drug metabolizing enzyme to be detected and the action target medicine, wherein the amplification primer can amplify a DNA sequence including a mutation site, and has at least 15 bases which are completely matched with the gene sequence aiming at the amplification primer at the 3 'end and a tag sequence of 10 bases (acgttggatg) at the 5' end so as to distinguish the amplification primer from an extension primer;
2) preparation of mass spectrum extension primer: designing an extension primer, wherein the extension primer is 15-28 bases in length, the 3' end of the extension primer is located at the last base in the extension direction of the drug-administration related mutation site, the extension primer only extends one base when an extension reaction occurs, and the extended base is the drug-administration related mutation site, wherein the molecular weight difference between a wild-type extension product and a mutant-type extension product of the extension primer is not less than 9Da (Da-Dalton), the molecular weight difference between the extension products of the drug-administration related mutation sites is not less than 30Da, and the primer is designed according to the principle that no dimer is formed between the primers, no hairpin structure is formed between the primers and an amplification product/extension product, and no mismatch occurs between the primers and a template;
in particular, the extension primer for mass spectrometric detection is designed according to the following principles: the molecular weights of the extension products are not less than 30Da, the molecular weight difference between the extension primers and the extension products is not less than 9Da, and the molecular weights of the extension primers and the extension products are 4300-8500 Da.
The information of the mutation sites of the genes related to the personalized medicine designed by the invention is shown in table 1, the amplification primers are shown in table 2, and the extension primers are shown in table 3.
TABLE 1 drug for lowering blood pressure related Gene mutation information
TABLE 2 PCR amplification primer sequences for the PCR amplifications
TABLE 3 Mass Spectrometry extension primers for the extension reaction
SEQ ID NO: Name of extension primer Primer sequence 5'→ 3' Extending bases
15 ADD1-UEP TGCTTCCATTCTGCC G
16 CYP2C9*3-UEP GGAACGAGGTCCAGAGATAC C
17 CACNA1C-UEP GTGGCCCTCCACAGTGCTGAC G
18 CYP2D6*10-UEP GCGCCAACGCTGGGCTGCACGCTAC G
19 SLCO1B1-UEP ATTCAACGAAGCATATTACCCATGAAC T
20 CYP3A5-UEP CCAAACAGGGAAGAGATA C
21 ADRB1-UEP GGAAGGCGCGCGCAGCAGAGCAGTC G
3) Preparation of a detection template: the mutation samples are tumor tissues (numbered as 2-A and 2-B) of 2 tumor patients examined in a hospital and 4 parts of normal human tissue samples (numbered as 3-A, 4-A, 5-A, 6-A and the like) to extract DNA (extracted by adopting a Tiangen DNA kit, and the operation method is extracted according to the kit specification);
4) performing PCR amplification by using the DNA extracted in the step 3) as a template and the PCR amplification primer in the step 1) to obtain a target sequence amplification product; see table 4 for PCR amplification reaction system; all reagents were purchased from the Agena Bioscience company, Inc., among others.
TABLE 4 PCR amplification reaction System
The PCR reaction condition was 94 ℃ for 2 minutes; denaturation at 94 ℃ for 30 seconds, annealing at 56 ℃ for 30 seconds, extension at 72 ℃ for 1 minute, and co-amplification for 45 cycles; final extension at 72 ℃ for 5 min. In this example, the DNA extracted in step 3) was used as a DNA template for PCR amplification. Meanwhile, sterile double distilled water was used as a negative control. And reacting and testing the control sample and the sample to be tested according to the same reaction process to verify the detection effectiveness.
5) Unreacted dNTPs contained in the amplification product obtained in step 4) are removed by SAP enzyme treatment. The SAP enzyme reaction system is shown in Table 5. All reagents were purchased from Agena Bioscience.
TABLE 5 SAP enzymatic reaction System
Reagent Volume/reaction
Water (HPLE grade) 1.53μl
SAP enzyme buffer 0.17μl
SAP enzymes 0.30μl
Obtaining PCR amplification product in step 4) 5.0μl
Total volume 7.0μl
SAP enzyme reaction conditions were 37 ℃ incubation for 40 minutes to remove residual dNTPs from the PCR amplification reaction; incubate at 85 ℃ for 5 minutes to inactivate SAP enzymes.
6) Using the amplification product obtained in the step 5) as a template, and connecting a base to the 3' end of the extension primer through an extension reaction by using the mass spectrum extension primer in the step 2), thereby obtaining an extension product. The extension reaction system is shown in Table 6. All reagents were purchased from Agena Bioscience.
TABLE 6 extension reaction System
Reagent Volume/reaction
Water (HPLE grade) 0.619μl
iPLEX buffer solution 0.200μl
iPLEXddNTP mixture 0.200μl
Extension primer mixtures in Table 3 *0.940μl
iPLEX enzyme 0.041μl
The product obtained in step (5) 7.00μl
Total volume 9.00μl
Wherein the mixture of extension primers is adjusted linearly according to the molecular weight of each primer (i.e., the amount of each primer used is calculated based on the molecular weight of each extension primer).
The extension reaction conditions were 94 ℃ for 30 seconds; denaturation at 94 ℃ for 5 seconds, annealing at 52 ℃ for 5 seconds, extension at 80 ℃ for 5 seconds, total amplification for 40 cycles, and annealing and extension in each cycle for 5 small cycles; final extension at 72 ℃ for 3 min.
7) The extension product obtained in step 6) was purified using a resin (purchased from Agena biosciences). To the extension product, 6mg of the resin and 16.00. mu.l of water were added and shaken vertically for one hour. After the reaction of the step, the resin is fully combined with cations in the reaction system, so that the reaction system is desalted. The purified product after the reaction is completed can be stored at 4 ℃ for several days or at-20 ℃ for several weeks. After the resulting purified product was centrifuged at 4000rpm for 5 minutes, the supernatant was taken and directly used for mass spectrometric detection.
8) The purified product was subjected to mass spectrometric detection on an Agena Bioscience MALDI-TOF mass spectrometer. The obtained mass peak patterns were analyzed by Typer4.0 analysis software (supplied by Agena Bioscience) to obtain typing results.
And (3) detection results: the mass spectrum peak analysis results are shown in FIGS. 1 to 3.
FIG. 1 is a mass spectrum peak diagram of ADD1-rs4961 detected by using DNA extracted from a sample as a template, and the analysis result is homozygous G, which shows that the curative effect on diuretic drugs is normal.
FIG. 2 is a mass spectrum peak diagram of ADD1-rs4961 detected by using DNA extracted from a sample as a template, and the analysis result is homozygous GT, which shows that the curative effect on diuretic drugs is enhanced.
FIG. 3 is a mass spectrum peak diagram of ADD1-rs4961 test using DNA extracted from sample as template, the analysis result is homozygous G, showing enhanced therapeutic effect on diuretic drugs.
Compared with the first-generation sequencing, the detection method and the detection kit have the advantages that the rapid turnaround time of one day is shorter; the reagent material consumption is simple, the experiment is operated in a flow process, and expensive reagents such as fluorescent dyes or probes are not needed; because the mass spectrometry technology directly detects the molecular weight (mass-to-charge ratio) of DNA, theoretically, only one copy of amplified DNA fragment can be amplified for identification, thereby having high sensitivity and smaller deviation compared with RT-PCR; the reaction can be carried out in a micro system, the capability of high multiplicity can be realized in a single reaction hole, the detection capability can reach 40 weight at most, and the detection efficiency is improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Sequence listing
<110> Kuo ran medical laboratory (Xuzhou) Co., Ltd
<120> detection method and kit for cardiovascular and cerebrovascular drug-related gene polymorphic sites
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 29
<212> DNA
<213> human genome (human genomic)
<400> 1
acgttggatg tgaactctgg ccggggcga 29
<210> 2
<211> 29
<212> DNA
<213> human genome (human genomic)
<400> 2
acgttggatg gtgatcgtgt gtaatgttc 29
<210> 3
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 3
acgttggatg tgtcacaggt cactgcatgg 30
<210> 4
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 4
acgttggatg ctacacagat gctgtggtgc 30
<210> 5
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 5
acgttggatg tcaacaacgc caacaacacc 30
<210> 6
<211> 29
<212> DNA
<213> human genome (human genomic)
<400> 6
acgttggatg gcccgtggcc ctccacagt 29
<210> 7
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 7
acgttggatg agtccacatg cagcaggttg 30
<210> 8
<211> 29
<212> DNA
<213> human genome (human genomic)
<400> 8
acgttggatg tggtggacct gatgcaccg 29
<210> 9
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 9
acgttggatg aatctgggtc atacatgtgg 30
<210> 10
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 10
acgttggatg ccaatggtac tatgggagtc 30
<210> 11
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 11
acgttggatg acccagctta acgaatgctc 30
<210> 12
<211> 30
<212> DNA
<213> human genome (human genomic)
<400> 12
acgttggatg gtaatgtggt ccaaacaggg 30
<210> 13
<211> 29
<212> DNA
<213> human genome (human genomic)
<400> 13
acgttggatg atcatctact gccgcagcc 29
<210> 14
<211> 29
<212> DNA
<213> human genome (human genomic)
<400> 14
acgttggatg ggtctccgtg ggtcgcgtg 29
<210> 15
<211> 15
<212> DNA
<213> human genome (human genomic)
<400> 15
tgcttccatt ctgcc 15
<210> 16
<211> 20
<212> DNA
<213> human genome (human genomic)
<400> 16
ggaacgaggt ccagagatac 20
<210> 17
<211> 21
<212> DNA
<213> human genome (human genomic)
<400> 17
gtggccctcc acagtgctga c 21
<210> 18
<211> 25
<212> DNA
<213> human genome (human genomic)
<400> 18
gcgccaacgc tgggctgcac gctac 25
<210> 19
<211> 27
<212> DNA
<213> human genome (human genomic)
<400> 19
attcaacgaa gcatattacc catgaac 27
<210> 20
<211> 18
<212> DNA
<213> human genome (human genomic)
<400> 20
ccaaacaggg aagagata 18
<210> 21
<211> 25
<212> DNA
<213> human genome (human genomic)
<400> 21
ggaaggcgcg cgcagcagag cagtc 25

Claims (10)

1. A detection method of the polymorphic locus of the cardiovascular and cerebrovascular medicine related gene is characterized in that the polymorphic locus of the cardiovascular and cerebrovascular medicine related gene comprises at least one of ADD1-RS4961, CYP2C9 3-RS1057910, CACNA1C-RS1051375, CYP2D6 10-RS1065852, SLCO1B1-RS4149056, CYP3A5-RS776746, ADRB1-RS 1801253;
the detection method comprises the following steps:
1) preparation of PCR amplification primers: the PCR amplification primer is designed according to the selected gene related to the individualized medication to be detected, and is specifically directed to each gene, the amplification primer can amplify a DNA sequence including a mutation site, the amplification primer has 17-25 basic groups which are completely matched with the gene sequence to which the amplification primer is directed at the 3 'end, and has a tag sequence at the 5' end so as to distinguish the amplification primer from an extension primer;
2) preparation of mass spectrum extension primer: the length of the mass spectrum extension primer is 15-28 bases, the 3' end of the mass spectrum extension primer is located at the last base of the mutation site of the related gene for personalized medicine in the extension direction, the extension primer only extends one base when in extension reaction, and the extended base is the mutation site of the related gene for personalized medicine;
3) preparation of a detection template: extracting DNA of a sample to be detected;
4) performing PCR amplification by using the DNA extracted in the step 3) as a template and the PCR amplification primer in the step 1) to obtain a target sequence amplification product;
5) removing unreacted dNTPs contained in the amplification product obtained in the step 4) by SAP enzyme treatment;
6) using the amplification product obtained in the step 5) as a template, and connecting a base to the 3' end of the extension primer through an extension reaction by using the mass spectrum extension primer in the step 2), thereby obtaining an extension product;
7) purifying the extension product obtained in step 6);
8) and (3) carrying out mass spectrum detection on the purified product on a mass spectrometer, and analyzing the obtained mass spectrum peak diagram through analysis software to obtain a typing result.
2. The method for detecting the polymorphic site of the cardiovascular and cerebrovascular drug-related gene according to claim 1, wherein in the step 1), the amplification primers have the same tag sequence at the 5' end.
3. The method for detecting the polymorphic site of the cardiovascular drug-related gene according to claim 2, wherein the tag sequence is acgttggatg.
4. The method for detecting the polymorphic sites of the genes related to cardiovascular and cerebrovascular medication according to claim 1, wherein the amplification primers in the step 1) comprise at least one pair of sequences from SEQ ID NO. 1 to SEQ ID NO. 14.
5. The method for detecting the polymorphic sites of the genes related to cardiovascular and cerebrovascular drugs according to claim 1, wherein the extension primers in step 2) comprise at least one pair of sequences from SEQ ID NO. 15 to SEQ ID NO. 21.
6. A detection kit for the polymorphic sites of related genes of cardiovascular and cerebrovascular medication is characterized by comprising a PCR amplification primer and a mass spectrum extension primer;
the PCR amplification primer is designed according to the selected gene related to the individualized medication to be detected, and is specifically directed to each gene, the amplification primer can amplify a DNA sequence including a mutation site, the amplification primer has 17-25 basic groups which are completely matched with the gene sequence directed to the amplification primer at the 3 'end, and has a tag sequence at the 5' end so as to distinguish the amplification primer from an extension primer;
the length of the mass spectrum extension primer is 15-28 bases, the 3' end of the mass spectrum extension primer is located at the last base in the extension direction of the mutation site of the gene related to the personalized medicine, the extension primer only extends one base when extension reaction occurs, and the extended base is the mutation site of the gene related to the personalized medicine.
7. The kit for detecting the polymorphic site of a cardiovascular and cerebrovascular drug-related gene according to claim 6, wherein the amplification primers have the same tag sequence at the 5' end.
8. The kit for detecting the polymorphic site of a gene related to cardiovascular and cerebrovascular administration according to claim 7, wherein the tag sequence is acgttggatg.
9. The kit for detecting the polymorphic sites of genes related to cardiovascular and cerebrovascular medication according to any one of claims 6 to 8, wherein the amplification primers comprise at least one pair of sequences from SEQ ID NO. 1 to SEQ ID NO. 14.
10. The kit for detecting the polymorphic sites of genes related to cardiovascular and cerebrovascular drugs according to any one of claims 6 to 8, wherein the extension primer comprises at least one pair of sequences from SEQ ID NO. 15 to SEQ ID NO. 21.
CN201910975111.7A 2019-10-14 2019-10-14 Method and kit for detecting polymorphism sites of cardiovascular and cerebrovascular drug-related genes Pending CN110592209A (en)

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Application publication date: 20191220